CN103207279A - Quick panda progesterone concentration determination method - Google Patents

Abstract

The invention discloses a quick panda progesterone concentration determination method. On the premise of no difference between detection results of urine samples, three hours for whole determination process of panda progesterone concentration is shortened to 1.5 hours by changing 2-hour incubation at conventional room temperature into 0.5-hour incubation at 37 DEG C, determination time is shortened by 50%, and precious time is earned for natural mating and artificial insemination in good time.

Description

The method for quick of giant panda progesterone concentration value

Technical field

The present invention relates to a kind of detection method that changes for progestational hormone in the oestrus female animal body, particularly relate to a kind of method for quick of giant panda progesterone concentration value.

Background technology

Giant panda is as the distinctive rare endangered wildlife of China, and its unique physilogical characteristics have attracted a large amount of scientific research personnel that it is studied.Female giant panda is single-shot feelings animals periodically, mostly be oestrus spring, the farrowing in autumn, in an oestrus, have only 1～3 day and accept natural mating.Under the stable breeding condition, have only the giant panda natural mating about 50%, there is the giant panda about half to finish breeding by artificial insemination.Judge in the shortest time whether giant panda ovulates, the pedestrian worker that goes forward side by side insemination is the key of Giant Panda in Reproduction success under the stable breeding condition.

In the Captive Giant Panda breeding in early days, topmostly judge whether giant panda oestruses the basis source of ovulation in the behavior observation of oestrusing of giant panda, comprise spirit, appetite, vulva color, nipple change, whether have baa, whether occur waiting to join posture and whether male giant panda mating desire etc. occurs to female giant panda.But because above method can't quantize fully, or some behavior all occurs in whole oestrus, thus can't judge very exactly whether giant panda ovulates, from but cause the low major reason of early stage Captive Giant Panda reproductive efficiency.

In actual giant panda breeding process, we need to collect behind the giant panda estradiol peak value sample more than 2 and could the basic time of determining that the estradiol peak value occurs according to detected urine sample progesterone value at least, at every turn from receiving that urine sample needs 3～5 hours at least to obtaining the result, often the time interval of our estradiol peak value of obtaining we to receive time of sample over and done with nearly 15 hours, this make us carry out in good time natural mating or the test-tube time very tightly short.

Summary of the invention

The technical problem to be solved in the present invention is a kind ofly to shorten the female giant panda urine examination time, ensure that giant panda has the method for quick of the giant panda progesterone concentration value of abundant natural mating time or artificial insemination time.

For solving the problems of the technologies described above, the method for quick of a kind of giant panda progesterone of the present invention concentration value may further comprise the steps:

(1) female giant panda is carried out A urine sample collection device, the freezing or stored refrigerated of the laggard row of mark on urine sample;

(2) with progestational hormone antibody CL425 96 hole ELISA Plate were spent the night bag by 6～48 hours; Described bag is that antibody is adsorbed on ELISA Plate, and purpose is in order to obtain the highest sensitivity, specificity, homogeneity and stability;

(3) will be divided into many aliquots with a urine sample; Use the hole selected good detection of 96 hole ELISA Plate; With concentration range 0, the 0.78to200pg/ hole, the progesterone standard items of totally 10 grades are done a series of grades and are doubly diluted; One aliquot urine sample is pressed 1:20,1:40,1:80,1:160,1:320 ... do a series of grades etc. equal proportion and doubly dilute, the urine sample of collecting when more oestrusing the peak near female giant panda, its extension rate is more high; The progesterone standard items that will dilute respectively then and urine sample join has in order successively wrapped by the detection of 96 good hole ELISA Plate with in the hole; At each detection of 96 hole ELISA Plate adding Horseradish peroxidase solution 0.05mL in the hole, after hatching 0.5 hour under 37 ℃, be NaCl, Tween20, H with principal ingredient ₂5 96 hole ELISA Plate of washing lotion washing of O;

(4) with the H of 0.04ml, concentration 0.5M ₂O ₂With 0.125ml, concentration be that the ABTS of 40mM joins at the bottom of the 12.5ml citric acid in the liquid, be mixed into chromophoric solution;

(5) each of 96 hole ELISA Plate after the washing detects with Kong Kongzhong and adds the chromophoric solution 0.1ml that has prepared, hatches under the room temperature after 30～60 minutes and reads absorbance value with microplate reader, and institute's value is this giant panda progesterone value;

(6) another aliquot urine sample is pressed 1:10～40 dilutions, be that the creatinine standard items of 0.1mg/ml are diluted to 0 with concentration, 6.25,12.5,25,50,100ug/ml, use the hole selected good detection of another 96 hole ELISA Plate then, the hole is used in urine sample and the rare detection that joins successively in order on the 96 hole ELISA Plate respectively of creatinine standard items of dilution, add 0.05mL in every detection in the hole again, concentration is NaOH and the 0.05mL of 0.75N, concentration is the picric acid of 0.4N, after at room temperature hatching 10～20 minutes, detect optical density value with microplate reader, institute's value is this giant panda urine sample creatinine value; When the creatinine value is lower than 0.1mg/mL, think sample by excess dilution, must adopt other to urinate little increment again and detect, when the creatinine value of detection urine sample is not less than 0.1mg/mL till;

(7) with this giant panda progesterone value of measuring divided by this giant panda creatinine value, the gained result is this giant panda progesterone concentration value, this concentration value is corrected value.

The method for quick of described a kind of giant panda progesterone concentration value, its each time the giant panda A urine sample collection device may further comprise the steps:

(1) cleans and drags dried beastly house ground;

(2) giant panda that will collect urine sample is put into beastly house, and the staff waits for, in case giant panda is discharged urine, immediately giant panda is put into other beastly house;

(3) gather the fresh urine sample in ground with clean injectors, the freeze pipe of packing into is carried out mark, puts into the freezing or refrigeration of refrigerator then immediately.

The method for quick of described a kind of giant panda progesterone concentration value, the Chinese of its Horseradish peroxidase are horseradish peroxidase; Its microplate reader the filter plate parameter be respectively 405nm, 450nm, 540nm, 490nm and 630nm; The Chinese of its ABTS is diamine salt chromophore; Its Tween20 is the potpourri of Tween 20 and the two Span 20s of a part of polyoxyethylene; In its measurement unit, mL is that mM, mg are that microgram, pg are that pik, nm are that nanometer, N are equivalent for milligram, ug for milliliter, M for mole, mM.

Be divided into 3 aliquots with per 1 part in 12 parts of female giant panda urine samples, under three kinds of different conditions, carry out detection and the correlation test of progesterone concentration value then respectively, the result as shown in Table 1 and Table 2:

The progesterone concentration value that three kinds of detection methods of table 1. obtain

Incubated at room 2 hours	Hatched 1 hour for 37 ℃	Hatched 0.5 hour for 37 ℃
			2.25	2.56	2.64
3.58	3.49	2.99
			4.50	5.03	4.79
4.46	4.08	5.32
			4.28	4.95	5.05
8.52	9.18	9.22
			7.30	11.25	7.83
10.95	11.26	9.03
			10.67	11.96	10.06
10.96	12.09	12.95
			15.46	21.29	14.03
17.24	19.11	13.39

The test of difference of the progesterone concentration value that table 2, three kinds of different detection methods obtain

According to table 1 and 2 as can be seen, 37 ℃ hatch 0.5 hour with traditional room temperature under hatch the result who drew in 2 hours and do not have difference.

The present invention is by changing into sample in 2 hours 37 ℃ and hatched 0.5 hour by hatching under traditional room temperature, make whole testing processes of giant panda progesterone concentration value shorten to 1.5 hours by original 3 hours, shortened 50% detection time, for valuable time has been striven in natural mating in good time and artificial insemination.

Description of drawings

The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.

Fig. 1 detects the front view of 96 hole ELISA Plate of progesterone value for the present invention;

Fig. 2 detects the front view in the ELISA Plate hole, 96 hole of creatinine value for the present invention.

Identify among the figure: S1 is to the detection hole of the progesterone standard items of the different dilute concentrations of S10 representative placement, T1 places the detection hole of different dilute concentration urine samples to the T26 representative, s1 is to the detection hole that the creatinine standard items of different dilute concentrations are placed in the s6 representative, and t1 places the detection hole of different dilute concentration urine samples to the t42 representative.

Embodiment

The present invention after cleaning and drying beastly house ground, collects need the female giant panda of urine sample and puts into beastly house during breed spring; The staff waits for then, in case giant panda is discharged urine, immediately giant panda is put into other beastly house, gathers the fresh urine sample in ground with clean injectors, and the freeze pipe of packing into is carried out mark, puts into the freezing preservation of freezer compartment of refrigerator or reefer stored refrigerated immediately.

To be divided into a plurality of aliquots with a urine sample, generally be divided into three aliquots at least.

When detecting the progesterone value, with progestational hormone antibody CL425 96 hole ELISA Plate were spent the night bag by 6～48 hours earlier; Then with concentration range 0, the 0.78to200pg/ hole, the progesterone standard items of totally 10 grades are done a series of grades and are doubly diluted, and are placed on the 96 hole ELISA Plate shown in Figure 1 S1 successively in the detection hole of S10; An aliquot urine sample is pressed 1:20,1:40,1:80,1:160,1:320 ... doing a series of grades etc. equal proportion doubly dilutes, the urine sample of collecting when more oestrusing the peak near female giant panda, its extension rate is more high, place on the 96 hole ELISA Plate T1 in the part or all of detection hole of T26 by the urine sample with dilution shown in Figure 1 then, the detection hole count of the shared 96 hole ELISA Plate of dilution urine sample is determined by the serial dilution multiple, extension rate is more high, and it is just more many to take the detection hole count; The back adds Horseradish peroxidase solution 0.05mL in each detection hole of 96 hole ELISA Plate shown in Figure 1 again, after hatching 0.5 hour under 37 ℃, is NaCl, Tween20, H with principal ingredient ₂5 96 hole ELISA Plate of washing lotion washing of O; H with 0.04ml, concentration 0.5M ₂O ₂With 0.125ml, concentration be that the ABTS of 40mM joins at the bottom of the 12.5ml citric acid in the liquid, be mixed into chromophoric solution; By shown in Figure 1, the chromophoric solution 0.1ml that adding has prepared in each the detection hole of 96 hole ELISA Plate after the washing is hatched under the room temperature after 30～60 minutes and is read absorbance value with microplate reader, and institute's value is this giant panda progesterone value.

When detecting the creatinine value, be earlier that the creatinine standard items of 0.1mg/ml are diluted to 0,6.25,12.5,25,50,100ug/ml with concentration, the creatinine standard items of dilution are placed on another 96 hole ELISA Plate shown in Figure 2 s1 in the detection hole of s6; Then with another aliquot urine sample in a certain dilution proportion in 1:10～40 ratios, be generally the 1:20 ratio, place on another 96 hole ELISA Plate t1 to the part or all of detection hole of t42 in by the urine sample with dilution shown in Figure 2, generally only need two detection holes; Each detects that to add 0.05mL, concentration in hole be that NaOH and 0.05mL, the concentration of 0.75N is the picric acid of 0.4N in 96 hole ELISA Plate shown in Figure 2 in the back again, after at room temperature hatching 15 minutes, detect optical density value with microplate reader, institute's value is this giant panda urine sample creatinine value; When the creatinine value is lower than 0.1mg/mL, think sample by excess dilution, must adopt other aliquot urine sample to detect again, when the creatinine value of detection urine sample is not less than 0.1mg/mL till.

At last, divided by this giant panda creatinine value, the gained result is giant panda progesterone concentration value with this giant panda progesterone value that measures, and this concentration value is corrected value.

The present invention is not limited to above embodiment, and the present invention also can be applied to the detection of other progestational hormone; T1 detects to T26 that t1 can place with a urine sample to t42 detection hole on hole and the shown in Figure 2 96 hole ELISA Plate on the 96 shown in Figure 1 hole ELISA Plate, also can place different urine samples, even place the urine sample of different female giant panda individualities.

Claims (2)

1. the method for quick of a giant panda progesterone concentration value may further comprise the steps:

(1) female giant panda is carried out A urine sample collection device, the freezing or stored refrigerated of the laggard row of mark on urine sample;

(2) with progestational hormone antibody CL425 with 96 hole ELISA Plate bags by 6～48 hours;

(3) with after progesterone standard items and the urine sample dilution, join successively in order respectively and wrapped by in the good ELISA Plate hole, 96 hole; In ELISA Plate hole, 96 hole, add Horseradish peroxidase solution, after hatching 0.5 hour under 37 ℃, wash 96 hole ELISA Plate with washing lotion;

(4) with H ₂O ₂Join at the bottom of the citric acid in the liquid with ABTS, be mixed into chromophoric solution;

(5) add the chromophoric solution prepared in the ELISA Plate hole, 96 hole after the washing, hatch under the room temperature after 30～60 minutes and read absorbance value with microplate reader, institute's value is this giant panda progesterone value;

(6) will join in the 96 hole ELISA Plate respectively in order successively after urine sample and the dilution of creatinine standard items, in ELISA Plate hole, 96 hole, add NaOH and picric acid again, after at room temperature hatching 10～20 minutes, detect optical density value with microplate reader, institute's value is this giant panda urine sample creatinine value;

(7) with this giant panda progesterone value of measuring divided by this giant panda creatinine value, the gained result is this giant panda progesterone concentration value.

2. the method for quick of a kind of giant panda progesterone concentration value according to claim 2, its each time the giant panda A urine sample collection device may further comprise the steps:

(1) cleans and drags dried beastly house ground;

(2) giant panda that will collect urine sample is put into beastly house, and the staff waits for, in case giant panda is discharged urine, immediately giant panda is put into other beastly house;

(3) gather the fresh urine sample in ground with clean injectors, the freeze pipe of packing into is carried out mark, puts into the freezing or refrigeration of refrigerator then immediately.

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